The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Plant tissue culture is now a proven technology for production of large numbers of genetically identical plants, however, its widespread application to various commercially important plants is generally restricted due to the following factors, which add up to the costs:                High labor input        Need for repeated sub-culturing        Poorly controlled in vitro environment        Limited rates of proliferation in vitro        Abnormal anatomical development, resulting in poor survival of plantlets upon transplantation to soil        A labor intensive hardening step before the plant can be established in fields.        Losses due to contamination        High costs incurred on                    power consumption during autoclaving, lighting and air-conditioning,            agar and sugar used in media preparation.                        
Thus, there is a need to develop efficient and dependable alternatives to achieve large scale micro propagation of plants, with minimum labor inputs. Partial automation of the micro propagation processes can serve as the most economically viable option. Liquid culture systems are necessary, considering ease in their automation. System automation could be brought about based on independently optimized processes, rather than automating the conventional procedures.
Bioreactors have traditionally been used for bacterial fermentation or for large scale production of secondary metabolites from plant cells. However, efforts to use the same for somatic embryogenesis/micro propagation could not succeed to the extent anticipated. The shear forces from fast impellers and foam formation in bubble aerated reactors caused most of the trouble (Heyerdahl et al., 1995). Many studies have been conducted to reveal the interactions between biological and physical parameters. Various guidelines for development of an automated system using bioreactors for plant tissue culture have been reviewed from time to time in the recent years (Aitken. Christie et al., 1995; Liu et al., 2003; Levin et al., 1997; Takayama and Akita, 1998; Ziv et al., 1998; Ziv, 2000). Depending on the type of explants, mode of propagation (organogenesis or embryogenesis) and the physiological and biochemical requirements of each of the stages of development, different approaches are followed for automation (Aitken-Christie, 1995). Various types of submerged culture bioreactors have been designed for generation of valuable secondary metabolites through intensive culture of organized tissues in liquid media (roots or transformed “hairy” roots, shoots, and embryos), or from undifferentiated single cells and aggregated cultures (Paek et al., 2001). Some of these include air-lift and bubble column-type bioreactors, balloon-type bubble bioreactors, stirred tank bioreactors, and ebb and flood type bioreactors. Suspension cultures of plant cells, which mimic microbial systems, constitute the primary route for secondary metabolite bioreactor operations, and the prime commercial route. These type of bioreactors have four major applications; (i) production of biomass (FIG. 2.1), (ii) production of secondary metabolites, (iii) production of enzymes, and (iv) biotransformation of exogenously added metabolites (which may be precursors in the pathway) (Leathers et al., 1994). Production of berberine from Thalictrum minus and Coptis japonica and shikonin from Lithospermum erythrorhizon are the two major examples of commercialized, bioreactor-based secondary metabolite production systems (Aitken-Christie, 1995). Fujita et al. (1982) reported industrialization of Lithospermum erythrorhizon cell suspension culture in 750-liter fermenter. Plant cell cultivation has been industrially accomplished up to a 75,000-liter bioreactor (Azechi et al., 1983; Rittershaus et al., 1989). Bioreactors devoted to mass propagation include systems for cultivation of cells, somatic embryos, or organogenic propagules (e.g., bulblets, corms, nodules, micro-tubers or shoot clusters) in liquid suspensions (Leathers et al., 1994). Balloon-type bubble bioreactors were developed for biomass production (Son et al., 1999a, 1999b). In most of these, the impeller of the conventional fermenter has been done away with, and both agitation and aeration are brought about by air spargers installed at their base. The shape of the vessel has also been frequently changed to shapes such as bubble shape and V-shape. Adaptation of air-lift and bubble-column bioreactors for propagation of shoot and bud clusters has provided a workable means for scale-up (Akita et al., 1994).
Though, several plants grow well in submerged culture in bioreactors, many plants fail to do so. Moreover, the survival of plantlets so obtained, is very low due to abnormal anatomy, and therefore, a separate hardening step is invariably required, unless the propagules are storage organs themselves, such as bulbs and tubers (Akita. and Takayama, 1988; Akita et al., 1994; Lim et al., 1998b; Seon et al., 2000; Son et al., 1999b; Takahashi et al., 1992; Yu et al., 2000). Because shoots are prone to hyperhydricity when grown in direct contact with liquid media, Ziv and co-workers (Ziv, 1990; Ziv, 1991a; Ziv and Ariel, 1991) used growth retardants to minimize leaf tissue development during the bioreactor proliferation stages of production. A 500 L aeration bioreactor used for shoots of Stevia rebaudiana, inoculated with 3 Kg fresh weight (FW) of primordia-like tissue, resulted in an exceptionally high yield (160 g/l), and subsequent re-establishment of shoots in soil (Akita et al, 1994).
Forays have also been made into development of bioreactors for non-submerged culture. Vessels have been developed that are provided with membrane rafts, or alternative supporting structure for holding the plant material in lieu of agar solidified medium (Leathers et al., 1994). The liquid nutrient medium may either be applied in the form of fine mist or may be filled in temporarily bringing about temporary immersion of the explants. Non-submerged culture type bioreactors are mainly for micro propagation and hairy root culture. Application of nutrient medium in the form of mist has been reported to be the most beneficial for culture of plantlets, as there is least amount of shear damage, and high surface area of the mist droplets result in high gaseous exchange. It has potential advantages in improving the diffusion of nutrient and gas in the region surrounding the cultured tissues. (Correl and Weathers, 1998; Correl and Weathers, 2000; Kim et al., 2001; Kim et al., 2002; Kim et al., 2003; Liu et al., 2003; Weathers and Giles, 1988). One of the earliest reports of a mist reactor with spray misting system was by Weathers and Giles (1988).
However, the work was not followed up to a scale-up stage due to various technical problems. In the same year Fox (1988) developed a system where the sprayer was replaced with an ultrasound transducer for generation of nutrient mist. In this system the transducer producing the fine nutrient mist was in direct contact with the nutrient medium. The life of the transducer was diminished by repeated autoclaving and experiments were often stopped because electrical components failed. Some of these problems were taken care of in the Acoustic Window Mist Reactor (AWMR) developed later by Chatterjee et al. (1997). They fabricated an acoustic window to separate the medium from the transducer using acoustically transparent epoxy resins initially, and later replaced it with an inexpensive polypropylene container. This system had been used for both, micro propagation: carnations (Correl and Weathers, 2000; Correl et al., 2000) as well as for hairy root culture (Woo et al., 1996). Acclimatization of the micro propagated plants could be brought about by a stepwise reduction in the relative humidity, resulting in good survival of plantlets ex vitro. Employing this technology, a bioreactor was developed by Waterford Equipment Company (New York). Herein, though initial growth of the plantlets was good, it did not last for long and necrosis set in finally, because of high concentration of residual salts on the surface of explants (Chatterjee et al., 1997).
On the other hand, application of mist in the form of spray was considered to be advantageous because of washing away of the toxins and less chances of accumulation of salts on the surface in high concentrations due to evaporation (Ibaraki and Kurata, 1991; Kurata et al., 1991). AWMR was modified by Chun et al. (1998) to develop a Modified Inner-Loop Mist Bioreactor (MILMB) of a capacity of 2.5 L. In this apparatus a concentric draught tube was provided inside the main culture vessel, which facilitated more uniform distribution of mist throughout the bioreactor volume as compared to the earlier AWMR. In an experiment, the shoots started turning brown after 25 days, most likely due to the same problem as described above (Chun et al., 2003). Woo et al. (1996) carried out hairy root culture in an AWMR, and compared the results with stirred tank and flask cultures. The increase in dry weight content in AWMR was just 4.76 times as compared to 12.9 times in stirred tank bioreactor and 21.4 times in flasks. This low growth rate in AWMR was probably due to limited nutrient supply of the air-carrier method. Because of its poor success, the prospects for scaling up of such a system for hairy root culture looked poor.
Another type of reactor used for micro propagation is chamber with provision for temporary immersion and forced ventilation, especially suitable for photoautotrophic culture. Kubota and Kozai (1992) used a vessel (2.6 L capacity) containing a multi-cell tray for keeping plants. Recently, Heo and Kozai (1999) developed a similar system using an even larger culture vessel (13 L capacity), with provision for CO2 enriched forced ventilation. In this system, plantlets were cultured photoautotrophically. However, one disadvantage of these large culture vessels was that growth of the cultured plantlets usually varied due to the non-uniform distribution of CO2 and other environmental factors in the culture headspace.
Similarly, Zobayed et al. (1999) developed an improved culture chamber (3.4 L capacity) with air distribution pipes to distribute the CO2-enriched air uniformly. However, in a larger (20 L) culture vessel, these pipes were unable to supply CO2-enriched air uniformly due to technical problems. Zobayed et al. (2000a) developed another scaled-up culture system with a larger (20 L) vessel made of acrylic. For uniform distribution of air, the chamber had a lower 2 mm high compartment with several vertical connecting tubes in between the upper and the lower chamber, directing the air flow from the air distribution chamber to the culture vessel headspace. The nutrient solution was supplied into the upper chamber with plants kept in plug trays to bring about temporary immersion. Its design did not allow flow of medium into the lower compartment. Drainage occurred along gravity.
RITA Vessel:
Yet another simple system was developed by CIRAD Biotrop, France to enable efficient culture of small sized explants such as somatic embryos. The system consisted of a small sized, two chambered main culture vessel with two ports for air supply and exit. Being small in size several vessels could be connected in a modular array, and the loss due to contamination was also restricted. The major advantage was its ability to only temporarily submerge the cultured plants, resulting in marked reduction in asphyxiation and tissue vitrification as compared to continuous immersion systems. Using this system, multiplication time could be reduced to upto half, the multiplication factor increased and the cost of production decreased to 1/10th (Teisson et al., 1996).
Notwithstanding the amount of innovations and research in automation of plant tissue culture, there are very few automated systems actually being used on a commercial scale at present due to various problems requiring attention (Aitken Christie et al, 1995 and Chu, 1994).
The in vitro environment is controlled to achieve different objectives for both, plant quality and production economy. Simple measures have proved to be effective in several cases.
Forced Air Circulation:
The typically stagnant culture vessel headspace (with high relative humidity, unfavorable gaseous composition and little air movement) was changed using altered vessel closures, forced introduction of sterile humidified air, etc. The plantlets so generated, had enhanced growth and superior ability to survive ex vitro (Kozai, 1991a; Kozai, 1991b; Kozai et al., 1992; Kubota and Kozai, 1992; Kozai, 1991c; McClelland and Smith, 1990; Smith and McClelland, 1991; Tanaka et al., 1992).
Recycle of Nutrient Medium:
Only some of the organic and inorganic nutrients added to the medium are absorbed by the cultures, and the residual nutrients including sugar are discarded together with the gelling agent after culture in almost all the cases. Recycling of nutrients, supporting materials and energy should become important in future tissue culture systems (Kozai and Smith, 1994).
Phototrophism:
It is well known that chlorophyllous cultures, in general, have a relatively high photosynthetic capacity and that these may grow faster in many cases under photoautotrophic conditions than under heterotrophic or photomixotrophic conditions, provided that the physical and chemical environment in the vessels are properly controlled for efficient photosynthesis (Pospisilova et al., 1992). Photoautotrophic cells are known to have well-developed and physiologically active chloroplasts, in contrast to heterotrophic cells (Hazarika, 2003). Control of in vitro environment also allows elimination of sugar from the culture medium (Hahn and Paek, 2001; Kozai, 1991a; Kozai, 1991b; Kozai, 1991c; Kozai et al., 1992; Kozai et al., 1996; Langford and Wainwright, 1987). Following are the potential advantages of photoautotrophic micropropagation (Fujiwara and Kozai, 1994; Kozai and Smith, 1994):    a) Growth and development of chlorophyllous cultures in vitro.    b) Physiological and/or morphological disorders are reduced.    c) Relatively uniform growth and development.    d) Procedures for rooting and acclimatization are simplified.    e) Application of growth regulators and other organic substances could be minimized thereby reducing mutations, and phenotypic variations.    f) Loss of cultures in vitro due to contamination can be reduced.    g) Larger vessels can be used with lowered risk of contamination (Kozai, 1991c).    h) Environment control of the vessel becomes easier due to reduced microbial contamination.    i) Asepsis in the vessel is not required as long as pathogens are excluded.    j) Automation, robotization and computerization become easier.
Introduction of forced ventilation, elevated CO2 and elimination of sugar from the media (Kozai, 1991a; Kozai, 1991c).
Provision of Adequate Growing Area:
The size and shape of the vessels/closures determine the limits of the growing space available to support plant growth. Besides, there may be some support at the bottom, for the plants. McClelland and Smith (1990) showed that explants routinely produced denser shoot cultures when grown in larger vessels. The quality of individual shoots was significantly better, shoot length in many species was enhanced, and size of individual leaves also increased with increase in size of the vessel. The rooting potential for micro-shoots produced in large sized vessels was also substantially improved, probably, in part, due to the enhanced leaf area and rooting cofactors present in these leaves as per their hypothesis. Photoautotrophic culture in large culture vessels with minimum risk of microbial contamination, along with forced ventilation is expected to reduce the labor costs by nearly 50% compared to conventional micropropagation systems (Kozai et al., 1999). Thus larger vessels as used in bioreactor systems are expected to promote the quality of plantlets cultured therein. However, Mackay and Kitto (1988) found that culture vessels that were excessively large also inhibited shoot length compared to the medium-sized vessels. Therefore, this parameter may need standardization for various plantlets to be propagated.
Thus, there is a need for an apparatus/bioreactor vessel that obviates the draw backs of the hitherto known prior art as detailed above.